National Standards of the People's Republic of China
gb 1886.355-2022
National standards for food safetyFood additivesSteviol glycosides
Foreword
This standard replaces GB 8270-2014 "National Food Safety Standard Food Additives - Steviol Glycosides".
Compared with GB 8270-2014, the main changes in this standard are as follows:
Modified standard scope;
Modified the molecular formula, structural formula and relative molecular weight
The index requirements and test methods for steviol glycoside content (on a dry basis) were modified
Modified the name of the "Residue on Ignition" indicator to "Ash".
The name of the indicator "Total Arsenic (as as)" has been changed to "Arsenic (AS)";
The test methods for lead (Pb) and arsenic (AS) have been added
The requirements of pH in the identification test were put into the physical and chemical indexes.
Scope
This standard is applicable to steviol glycosides, a food additive obtained from stevia (Stevia rebaudiana bertoni) leaves, which are extracted and refined. Known glycosides include steviosides, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside N, rebaudioside O, Durkoside A, sweet tea glycosides, and steviol disaccharides.
Molecular formula, structural formula, and relative molecular weight
2.1 Molecular formula of 13 glycosides.
Steviolin: C38H60O18
Rebaudioside A: C44H70O23
Rebaudioside B: C38 H60O18
Rebaudioside C: C44H70O22
Rebaudioside D: C50H80O28
Rebaudioside E: C44H70O23
Rebaudioside F: C43H68O22
Rebaudioside M: C56H90O33
Rebaudioside N: C56H90O32
Rebaudioside O: C62H100O37
Ducoside A: C38H60O17
Sweet tea glycosides: C32H50O13
Stevia disaccharides: C32H50O13
2.2 Structural formula of 13 glycosides.
The compound names, R1 substituents and R2 substituents of the 13 glycosides are shown in Table 1. Steviol (r1=r2=h) is a steviol aglycone.
2.3 Relative molecular weights of 13 glycosides.
Steviolin: 80487 (by 2018 international relative atomic mass).
Rebaudioside A: 96701 (by 2018 international relative atomic mass).
Rebaudioside B: 80487 (by 2018 international relative atomic mass).
Rebaudioside C: 95101 (by 2018 international relative atomic mass).
Rebaudioside d: 1 12915 (by international relative atomic mass in 2018).
Rebaudioside E: 96701 (by 2018 international relative atomic mass).
Rebaudioside F: 93699 (by international relative atomic mass in 2018).
Rebaudioside m: 1 29129 (by 2018 international relative atomic mass).
Rebaudioside n: 1 27529 (by 2018 international relative atomic mass).
Rebaudioside O: 1 43744 (by international relative atomic mass 2018).
Ducoside A: 78887 (by 2018 international relative atomic mass).
Sweet tea glycosides: 64273 (by international relative atomic mass 2018).
Steviol disaccharides: 64273 (by international relative atomic mass 2018).
Technical requirements
3.1 Sensory requirements.
Sensory requirements should be in accordance with Table 2.
3.2. Physical and chemical indicators.
The physical and chemical indicators should be in accordance with the provisions of Table 3.
Appendixa
Test Method
a.1General provisions
The reagents and water used in this standard refer to the pure reagents for analysis and the ** water specified in GB T 6682 when no other requirements are indicated. The standard titration solution, standard solution for impurity determination, preparations and products used in the test are prepared in accordance with the provisions of GB T 601, GB T 602 and GB T 603 when no other requirements are indicated. The solution used in the test refers to the aqueous solution when the solvent is not indicated.
a.2Identification test
a.2.1. Steviol glycoside chromatographic test.
In the steviol glycoside content test, the chromatographic peaks of the 13 glycosides in the chromatogram of the sample solution should correspond to the mixed standard solution.
a.3Steviol glycoside contentOn a dry basisDetermination of
a.3.1. Reagents and materials.
a.3.1.1 Acetonitrile: chromatographically pure.
a.3.1.2 Sodium phosphate monobasic: chromatographically pure.
a.3.1.3 Phosphoric acid: chromatographically pure.
a.3.1.4 Water: Class 1 water specified in GB T 6682.
a.3.1.5 Aqueous acetonitrile solution: The volume ratio of acetonitrile to water is 30:70.
a.3.1.6. Sodium phosphate buffer (pH 2.)6): Weigh 120 g sodium phosphate monobasic (NaH2PO4), dissolved in 800 ml of water, adjust pH to 2 with phosphoric acid6。
a.3.1.7 Rebaudioside A standard: Rebaudioside A content (mass fraction, on a dry basis) 990%。
a.3.1.8 Rebaudioside D Standard: Rebaudioside D content (mass fraction, on a dry basis) 950%。
a.3.1.9 11 other glycoside standards: stevioside, rebaudioside B, rebaudioside C, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside N, rebaudioside O, Durkoside A, sweet tea glycosides, and steviol disaccharides, or use a mixture of 13 glycosides.
a.3.2 Instruments and equipment.
High Performance Liquid Chromatograph: Equipped with a UV detector, or other equivalent detector.
a.3.3 Refer to chromatographic conditions.
a.3.3.1 Column: C18 reversed-phase column, 250 mm 46 mm, particle size 5 m;or other equivalent columns.
a.3.3.2. Mobile phase gradient elution conditions (the proportion can be adjusted according to the actual situation): see Table a1。
a.3.3.3. Mobile phase flow rate: 08 ml/min。
a.3.3.4 Detection wavelength: 210 nm.
a.3.3.5 Injection volume: 2 l 10 l.
a.3.3.6. Column temperature: 40.
a.3.4 Analytical steps.
a.3.4.1. Preparation of mixed standard solution.
Weigh an appropriate amount of Rebaudioside A, Rebaudioside D, Steviol Rebaudioside B, Rebaudioside C, Rebaudioside E, Rebaudioside F, Rebaudioside M, Rebaudioside N, Rebaudioside O, Dukeside A, Sweet Tea Glycoside and Steviol Disaccharide Standard (or 13 kinds of glycoside mixed standards), put them in the same volumetric flask, and completely dissolve them with acetonitrile aqueous solution to obtain a mixed standard solution. The mixed standard solution was used to determine the relative retention time of the 13 glycosides.
a.3.4.2. Preparation of standard solutions.
Weigh 50 mg、10.0 mg、20.0 mg、25.0 mg of rebaudioside A standard and 25 mg、5.0 mg、10.0 mg、12.5 mg of rebaudioside D standard to the nearest 01 mg, respectively placed in a 25 ml volumetric flask, dissolved in acetonitrile aqueous solution and diluted to the scale, respectively, to obtain a concentration of 200 mg L, 400 mg L, 800 mg L, 1 000 mg L of rebaudioside A standard solution and 100 mg L, 200 mg L, 400 mg L, 500 mg L of rebaudioside D standard solution.
a.3.4.3. Preparation of sample solution.
Weigh 250 mg±5.0 mg specimen (dry basis) to the nearest 01 mg, placed in a 50 ml volumetric flask, dissolved in aqueous acetonitrile solution, diluted to the scale to obtain a sample solution.
a.3.5 Assay.
a.3.5.1. Drawing of the standard curve.
In a3.3. Under reference chromatographic conditions, chromatographic analysis was performed on 200 mg L, 400 mg L, 800 mg L, 1 000 mg L of Rebaudioside A standard solution and 100 mg L, 200 mg L, 400 mg L, 500 mg L of Rebaudioside D standard solution, respectively, and the corresponding peak area values of each concentration of the standard solution were recorded. The peak area value of rebaudioside A or rebaudioside D in the chromatogram of the standard solution was used as the y-axis, and the corresponding solution concentration (mg L) was used as the x-axis to plot the standard curve of rebaudioside A and rebaudioside D (forced to cross the origin), and the linear function slope (Ka) and rebaudioside D linear function slope (KD) were obtained from the standard curve, respectively.
a.3.5.2. Content determination.
In a3.3. Under reference chromatographic conditions, chromatographic analysis was performed on the mixed standard solution and the sample solution respectively. The chromatogram of the sample solution is compared to the chromatogram of the mixed standard solution (see Appendix B) to determine the corresponding peaks for each component in the sample solution chromatogram. The peak areas of Rebaudioside A, Rebaudioside D, steviolin, Rebaudioside B, Rebaudioside C, Rebaudioside E, Rebaudioside F, Rebaudioside M, Rebaudioside N, Rebaudioside O, Durk Glycoside, Sweet Tea Glycosides, and Stevia Disaccharides were recorded.
a.3.6 Result Calculation.
Mass fraction of 8 glycosides (on a dry basis) w;Press formula (a.)1) Calculation.
Where: i-S, A, B, C, F, DA, RU, SB, corresponding to steviol rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside F, Durk glycoside A, sweet tea glycosides, steviol disaccharides;
The ratio of the formula to the fi-i component and rebaudioside a: 100 (Rebaudioside A.)83 (stevioside. 83 (rebaudioside b),
0.98 (Rebaudioside C.)97 (Rebaudioside F.)82 (Ducoside A.)66 (sweet tea glycosides. 66 (steviol disaccharide);
AI – the peak area value of component I in the chromatogram of the sample solution;
ka—— a.3.5.1 Slope of the linear function of rebaudioside a;
c – the concentration of the sample solution in milligrams per liter (mg l).
Mass fraction of 5 glycoside contents (on a dry basis) by formula (a.).2) Calculation.
Where: i-d, e, m, n, o, corresponding to rebaudioside D, rebaudioside E, rebaudioside m, rebaudioside N, rebaudioside O;
The ratio of the formula to the rebaudioside D of the Fi-I component: 100 (Rebaudioside D.)86 (Rebaudioside E.)14 (rebaudioside.)
m.13 (Rebaudioside n.)27 (rebaudioside O);
AI – the peak area value of component I in the chromatogram of the sample solution;
kd——a.3.5.1 Slope of the linear function of rebaudioside d;
c – the concentration of the sample solution in milligrams per liter (mg l).
By Eq. (a.).1) and Eq. (a.)2) The contents of 13 components s, a, b, c, f, da, ru, sb, d, e, m, n and 0 are calculated, and the sum of the contents of each component is the steviol glycoside content in the sample.
a.4 phDetermination of
a.4.1 Instruments and equipment.
Acidity meter. a.4.2. Reagents and materials.
Weigh 1 g of the sample, dissolve it in 100 ml of water with carbon dioxide removed, and determine the pH of the sample solution with an acidity meter.
a.5Determination of methanol and ethanol
a.5.1. Reagents and materials.
a.5.1.1 Methanol: chromatographically pure.
a.5.1.2 Ethanol: chromatographically pure.
a.5.1.3 Water: Class 1 water according to GB T 6682.
a.5.2 Instruments and equipment.
Gas chromatograph: Equipped with a hydrogen flame ionization detector (FID) and headspace injector.
a.5.3 Refer to chromatographic conditions.
a.5.3.1 Column: bonded polyethylene glycol fused silica capillary column (column length is 30 m, column inner diameter is 0.).25 mm with a film thickness of 025 m), or other equivalent column.
a.5.3.2. Carrier gas: nitrogen ( purity 9999%)。
a.5.3.3. Carrier gas flow: 50 ml/ min。
a.5.3.4. Column temperature: 40 for 5 min, 10 min to 120 min, 2 min for 16 min.
200 for 5 min.
a.5.3.5. Injection port temperature: 200 °C.
a.5.3.6. Detector temperature: 250.
a.5.3.7 Injection volume: 1 ml.
a.5.3.8 shunt ratio: 1:50.
a.5.4. Refer to headspace conditions.
a.5.4.1. Headspace bottle temperature: 80.
a.5.4.2. Equilibration time of headspace bottle: 30 min.
a.5.5 Analytical steps.
a.5.5.1. Preparation of blank solution.
Pipette 2 ml of water, place in a headspace bottle, quickly press the cap tightly and set aside.
a.5.5.2. Preparation of standard solutions.
a.5.5.2.1. Preparation of methanol standard solution.
Weigh 01 g methanol, accurate to 0001 g, diluted with water, transferred to a 1 000 ml volumetric flask, added to the scale with water, shaken well, and 100 mg l of methanol standard stock solution was obtained. The solution is formulated into a series of concentrations of 2Methanol standard solutions of 5 mg L, 5 mg L, 10 mg L, 20 mg L, and 50 mg L. Pipette 2 ml of each of the above series of concentration solutions, place them in headspace bottles, quickly press the caps, and set aside.
a.5.5.2.2. Preparation of ethanol standard solution.
Weigh 01 g ethanol, accurate to 0001 g, diluted with water, transferred to a 100 ml volumetric flask, added to the scale with water, shaken well, and 1 000 mg l of ethanol standard stock solution was obtained. The solution was prepared into a series of ethanol standard solutions at concentrations of 50 mg L, 100 mg L, 200 mg L, 500 mg L, and 750 mg L. Pipette 2 ml of each of the above series of concentration solutions, place them in headspace bottles, quickly press the caps, and set aside.
a.5.5.3. Preparation of sample solution.
Weigh 10g specimen, accurate to 0001 g, dissolved in water and transferred to two 10 ml volumetric flasks, sonicated at room temperature for about 3 min, diluted to the scale with water, and shaken. Pipette 2 ml of the solution and place it in a headspace bottle, quickly press the cap tightly and set aside.
a.5.6Assay
In reference to operating condition a5.3 and a5.4. The blank solution, the standard series solution and the sample solution were measured respectively, and the peak area value of methanol or ethanol was recorded. The peak area value of methanol or ethanol in the chromatogram of the standard series solution was used as the y-axis, and the corresponding solvent concentration (mg L) was used as the x-axis, and the standard curve of methanol or ethanol was obtained. According to the peak area value of methanol or ethanol in the chromatogram of the sample solution, the concentration of methanol or ethanol in the sample solution (mg l) was obtained from the standard curve.
a.5.7Result calculation
The amount of methanol or ethanol in the sample w, measured in milligrams per kilogram (mg kg), according to the formula (a.).3) Calculation.
Where: c - the concentration of methanol or ethanol in milligrams per liter (mg l) of the sample solution obtained from the standard curve;
v - the volume of the sample solution, in milliliters (ml);
m – the mass of the specimen in grams (g).
*From: